Insulin Regulation of Calpain Action. The long-range goal of this project is to understand the physiological role and regulation of calpain in disease processes. The immediate goal is to determine the contribution of phospholipids in the regulation of calpain activity by insulin. The P.I. has discovered that exposure of 3T3-L1 adipocytes to insulin (EC50 = 350 pM) for 4 h activates m-calpain leading to the degradation of the insulin receptor substrate 1 (IRS-1). Calpain is a widely distributed family of closely related calcium-dependent proteases normally found inactive throughout the cytosol of cells of nearly all tissues. Milli-calpain requires calcium ion concentrations near 1 mM. Calpain activation often occurs in close association with elevations in calcium ion concentration as seen where calpain action leads to damage to tissue proteins. However, calcium levels do not reach levels high enough to activate milli-calpain. Therefore, other factors must be operative. Insulin stimulation of cells causes the production of membrane-bound phosphatidylinositol (PtdIns) 3-phosphates. Calpain binds these phospholipids in vitro leading to a reduced requirement for calcium ion. These data have led to the hypothesis that the insulin stimulation of calpain degradation of IRS-1 requires the interaction of calpain and IRS-1 with acidic phospholipids and is modulated by calpain activators and inhibitors. Three Specific Aims have been designed to test this hypothesis. Aim 1 is to characterize the lipid-binding site of the 30-kDa subunit of calpain and to determine if it is required in the insulin stimulation of calpain activity. Aim 2 is to characterize the lipid-binding properties of the pleckstrin homology domain of IRS-1 and to determine its role in calpain degradation or IRS-1. Aim 3 is to determine if insulin stimulation affects expression and subcellular distributions of calpain activator and inhibitor proteins. Accomplishing these goals will contribute our understanding of several disease processes that involve calpain, for example Alzheimer's Disease and muscular dystrophy. Furthermore, these studies may reveal a new role of calpain in insulin resistance syndromes that are due to persistent elevations in insulin.